The present invention relates to a novel class of peptide compounds that specifically block class E voltage-gated calcium channels and to methods for blocking such channels. The invention also, relates to therapeutic treatments, such as treatment of convulsive disorders, using the compounds.
Banga, A. K., Therapeutic Peptides and Proteins. Technomic Publishing Inc., Lancaster, Pa. (1995).
Biel, M., et al., FEBS Lett. 269:409-412 (1990).
Bruckner, H., et al., J. Chromatog. 476:73-82 (1989).
Carbone, E., et al., Pflugers Arch. 416:170-179 (1990).
Cohen, C. J., et al., Mol Pharmacol. 42:947-951 (1994).
Ellinor, P. T., et al., Nature 372(6503):272-275 (1994).
Ellis, et al., Science 241 (4873):1661-1664 (1988).
Ertel, E. A., et al., Biochemistry 33:5098-5108 (1994).
Forti, L., et al., J. Neurosci. 14:5243-5256 (1994).
Frohman, M. A., et al., Proc. Natl. Acad. Sci. USA 85:8998-9002 (1988).
Frohman, M. A., in PCR PROTOCOL: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, New York, N.Y., p. 28 (1990).
Fujita, Y., et al., Neuron 10:585 (1993).
Hamil, O. P., et al., Pflugers Archiv. 391: 85-100 (1981).
Grynkiewicz, G., et al., J. Biol. Chem. 260:3440-3450 (1985)
Hagiwara, K., et al., Biomed. Res. 11:181-186 (1990).
Hamill, O. P., et al., Pflugers Arch. 391:85-100 (1981).
Hawke, D., and Yuan, P., Appl. Biosystems User Bull. 28:1-8 (1987).
Hillyard, et al., Neuron 9:69-77 (1993).
Horne, W. A., et al., PCT/US94/08589 (WO 95/04144) (1995).
Jackson, H. C. and Scheideler, M. A. Psychopharmacology 126: 85-90, 1996.
Lampe, R. A., et al., Mol. Pharmacol. 44:451-460 (1993).
Lievano, A., et al., Am. J. Physiol. 267 (Cell Physiol.):C411-C424 (1994).
Llinas, R., et al., Trends Neurosci. 15:351-354 (1992).)
Maniatis, T., et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).
Mikami, A. et al., Nature 340:230-233(1989).
Mori, Y., et al., Nature 350:398-402 (1991).
Newcomb, R., et al., Biochemistry 34:8341-8347 (1995).
Newcomb, R., et al., LC-GC Mag. Sep. Sci. 7: 570-578 (1989).
Palma, A., et al., Soc. Neurosci. Abstr. 20(1-2) (1994).
Perez-Reyes, E., et al., Nature 340:233-236 (1989)
Piser, T. M., et al., Mol. Pharmacol. 48:131-139 (1995).
Premack, A. B., et al., J. Immunol. 152:5226-5240 (1994).
Rae, J., et al., J. Neurosci. Meth. 87:15 (1991).
Ramaswami, M., et al., Mol. Cell. Neurosci. 1:214-223 (1990).
Randall, A., and Tsien, R. W., J. Neurosci. 15:2995-3012 (1995).
Sather, W. A., et al., Neuron 11:291-303 (1993).
Smith, P. K., et al., Anal. Biochem. 150:76 (1985).
Snutch, T. P., et al., Proc. Natl. Acad. Sci. 87:3391-3395 (1990).
Soong, T. W., et al., Science 260:1133-1136 (1993).
Stea, A., et al., Proc. Natl. Acad. Sci. 91:10576-10580 (1994).
Swartz, K. J., and MacKinnon, R., Neuron 15:941-949 (1995).
Tanabe, T., et al., Nature 328:313-318 (1987).
Tarr, G. E., et al., Proc. Natl. Acad. Sci. USA 80:6552-6556 (1993).
Tempel, B. L., et al., Nature 332:.837-839 (1988).
Tsien, R. W., et al., Trends Neurosci. 12:349-354
Venema, V. J., et al., J. Biol. Chem. 267:.2610-2615 (1992).
Wang, J., et al. J. Physiology, in press.
Williams, M. E., et al., Neuron 8:71-84 (1992).
Yaksch, T. L., and Rudy, T. A., Physiol. Behav. 17:1031-1036 (1976).
Voltage-gated calcium channels are present in neurons, and in cardiac, smooth, and skeletal muscle and other excitable cells. These channels are known to be involved in membrane excitability, muscle contraction, and cellular secretion, such as in exocytotic synaptic transmission. In neuronal cells, voltage-gated calcium channels have been classified by their electrophysiological as well as by their biochemical and pharmacological properties. More recently, further classification has been made based on the molecular biology of the channels. Calcium channels are generally classified according to their electrophysiological properties as Low-voltage-activated (LVA) or High-voltage-activated (HVA) channels. HVA channels are currently known to comprise at least three groups of channels, known as L-, N- and P/Q-type channels. These channels have been distinguished one from another electrophysiologically as well as bio-chemically on the basis of their pharmacology and ligand binding properties. Thus, dihydropyridines, diphenyl-alkylamines and piperidines bind to the alpha1 subunit of the L-type calcium channel and block a proportion of HVA calcium currents in neuronal tissue, which are termed L-type calcium currents. N-type calcium channels are sensitive to omega conopeptides, but are relatively insensitive to dihydropyridine compounds, such as nimodipine and nifedipine. P/Q-type channels, on the other hand, are insensitive to dihydropyridines, but are sensitive to the funnel web spider toxin Aga IIIA.
R-type calcium channels, like L-, N-, P- and Q-type channels, are activated by large membrane depolarizations, and are thus classified as high voltage-activated (HVA) channels. R-type channels are insensitive to dihydropyridines and omega conopeptides, but, like P/Q, L and N channels, are sensitive to the funnel web spider toxin AgaIVA. Immunocytochemical staining studies indicate that these channels are located throughout the brain, particularly in deep midline structures (caudate-putamen, thalamus, hypothalamus, amygdala, cerebellum) and in the nuclei of the ventral midbrain and brainstem. This channel is thought to reside primarily in neuronal cell bodies and dendrites, where it contributes to cellular electrical activity. There is now also evidence that R-type channels may be localized on pre-synaptic nerve terminals.
The molecular complex comprising neuronal voltage-sensitive calcium channels consists of a central xcex11 subunit, an xcex12/xcex4 subunit, a xcex2 subunit and a 95 kD subunit. Molecular genetic studies have revealed at least five mRNA classes that code for xcex11 subunits, designated A, B, C, D, and E. These correspond to the P/Q-type (xcex11A), N-type (xcex11B), L-type (xcex11C, xcex11D), and R-type (xcex11E) voltage-gated channels, as defined by electrophysiological studies. (Snutch et al. 1990, Soong et al. 1993, Tsien et al. 1991; Biel et al. 1990, Mikami et al. 1989, Perez-Reyes et al. 1989, Tanabe et al., Williams et al., 1992; Fujita et al. 1993; Mori et al. 1991, Sather et al. 1993, Stea et. al. 1994 Forti et al. 1994, Randall and Tsien 1994). The class E voltage-gated calcium channel encompasses currents characterized electrophysiologically as R-type and G2 currents.
There are no known specific or selective ligands for the Class E or R-type neuronal calcium channel. Although the spider peptide omega-Aga IIIA antagonizes this channel, it also potently blocks N, P/Q- and L-type calcium currents (Cohen et al. 1993, Ertel et al. 1994) and therefore lacks specificity. The lack of specific ligands for the channel has heretofore impeded elucidation of its role(s) in neuronal function. Table 1 summarizes antagonists of the various subtypes of voltage-gated calcium channels referred to herein.
In view of the importance of specific calcium channels in neuronal function, it would be useful to identify pharmacological agents that specifically block the class E calcium channel. The present invention is based on the discovery of a new class of peptides that selectively block this channel. This class of compounds is exemplified herein by the novel HG peptides described herein that are derived from peptides originally isolated from H. gigas. 
The present invention relates to a novel class of peptides that selectively block class E calcium channels. More specifically, such peptides, generally referred to as class E voltage-gated calcium channel blocking peptides herein, are capable of blocking class E voltage-sensitive calcium channels at a concentration that is about 10-50, and more preferably no more than 10-20, times the concentration of HG-1 peptide (SEQ ID NO: 1) required to block such channels. The peptides may be further characterized, in another embodiment, by an inability of the particular peptide to block by at least 50% L-type, T-type, P/Q-type or N-type calcium channels, as described herein, at a molar concentration that is about 10 times the molar concentration required to half maximally block a class E voltage-gated calcium channel.
In a preferred embodiment, the peptide takes the form: V1-SEQ ID NO: 19-X2X3X4-SEQ ID NO: 20-X5-LGC-X6X7X8X9-S-X10-SEQ ID NO: 10-X11X12-T-X13X14X15 where V1 is GVDKX1G or deletion, X1 being A or P; X2 is S or E, X3 is V or K, X4 is D or N, X5 is R or K, X6 is H or K, X7 is S or D, X8 is I or L, X9 is F or L, X10 is Y or deletion, X11 is D or E, X12 is L or V, X13 is F or G, X14 is S or E or deletion, and X15 is F or D or deletion.
In another preferred embodiment, the peptide has the form: SEQ ID NO: 16-X1-FGGC-X2X3X4-SEQ ID NO: 17-X5X6X7X8X9X10-SEQ ID NO: 18-X11-TFSD, wherein X1 is selected from Class V; X2 is selected from Class II; X3 is selected from Class IV or V; X4 is selected from Class III; X5 is selected from Class III or Class IV or deletion; X6 is selected from Class IV or V; X7 is selected from Class II or IV; X8 is selected from Class II or V; X9 is selected from Class VI; X10 is selected from Class II or III; and X11 is selected from Class II, V or VI. In further preferred embodiment, the variable positions in the above composite peptide assume the following substituents: X1=M or L; X2=S or T; X3=V, K or R; X4=N or D; X5=K, Q or a deletion; X6=H, R, or L; X7=S or K; X8=L or G; X9=F or Y; X10=S or N; and X11=L, F, or G. In still another preferred embodiment, the peptide is any of SEQ ID NO: 1 (HG-1), SEQ ID NO: 8 (HG-8), SEQ ID NO: 13 (R9), SEQ ID NO: 14 (R11), and SEQ ID NO: 15 (SNX-629).
In another embodiment, the invention includes isolated polynucleotides, comprising, a sequence of nucleotides that encode a peptide selected from the peptides having the sequences defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In another embodiment, the polynucleotide is selected from nucleotides having sequences defined by SEQ ID NO: 23 to SEQ ID NO: 43.
In still a further embodiment, the isolated polynucleotide comprises a sequence of nucleotides that encodes a peptide having the sequence: V1-SEQ ID NO: 19-X2X3X4-SEQ ID NO: 20-X5-LGC-X6X7X8X9-S-X10-SEQ ID NO: 10-X11X12-T-X13X14X15 where V1 is GVDKX1G SEQ ID NO: 51 or deletion, X1 being A or P; X2 is S or E, X3 is V or K, X4 is D or N, X5 is R or K, X6 is H or K, X7 is S or D, X8 is I or L, X9 is F or L, X10 is Y or deletion, X11 is D or E, X12 is L or V, X13 is F or G, X14 is S or E or deletion, and X15 is F or D or deletion.
In another embodiment, the isolated polynucleotide encodes a peptide having the sequence SEQ ID NO: 16-X1FGGC-X2X3X4-SEQ ID NO: 17-X5X6X7X8X9X10-SEQ ID NO: 18-X11-TFSD, wherein X1 is selected from Class V; X2 is selected from Class II; X3 is selected from Class IV or V; X4 is selected from Class III; X5 is selected from Class III or Class IV or deletion; X6 is selected from Class IV or V; X7 is selected from Class II or IV; X8 is selected from Class II or V; X9 is selected from Class VI; X10 is selected from Class II or III; and X11 is selected from Class II, V or VI.
In a related aspect, the invention includes a method of inhibiting seizures in a subject. The method includes administering to the subject a pharmaceutically effective dose of an HG peptide capable of blocking a class E voltage-gated calcium channel at a concentration that is at most about 10-50 times the concentration of HG-1 peptide (SEQ ID NO: 1) required to block said channel.
In a related embodiment, the peptide used in the anticonvulsive treatment method is further characterized by inability to block by at least 50% L-type, T-type, class A (P/Q type) or class B (N-type) calcium channels at a concentration that is at most about 10-times the concentration required to half maximally block said class E Voltage-gated calcium channel. A preferred peptide for use in this method has the sequence: V1-SEQ ID NO: 19-X2X3X4-SEQ ID NO: 20-X5-LGC-X6X7X8X9-S-X10 SEQ ID NO: 10-X11X12-T-X13X14X15 where V1 is GVDKX1G (SEQ ID NO: 51) or deletion, X1 being A or P; X2 is S or E, X3 is V or K, X4 is D or N, X5 is R or K, X6 is H or K, X7 is S or D, X8 is I or L, X9 is F or L, X10 is Y or deletion, X11 is D or E, X12 is L or V, X13 is F or G, X14 is S or E or deletion, and X15 is F or D or deletion. Preferred peptides for use in this method include SEQ ID NO: 1 and SEQ ID NO: 15.
In a further related aspect, the invention includes methods of inhibiting release of neurohypophysial hormones, such as oxytocin, into circulation. Such a method can be employed, for example, to prevent premature labor or to inhibit the let-down response of lactation. The method includes administration of a class E channel blocking HG peptide in an appropriate pharmaceutical excipient. In addition to the HG-peptides which block class E channels, as discussed herein, this treatment paradigm may include administering an L-type calcium channel blocker, an N-type calcium channel blocker or a P/Q type calcium channel blocking agent. An effective therapeutic regimen, in this context, is one in which premature labor is abolished. Such a regimen may also be used to inhibit the lactation let-down response in a lactating female.
The invention also includes methods of selecting compounds for use in the foregoing treatment methods (inhibition of prolactin release, anticonvulsant activity). The method includes testing a compound for ability to selectively block class E calcium channels in neuronal tissue, and selecting the compound it blocks calcium currents through said class E calcium channels at a concentration that is no more than about 10-50 times a concentration of HG-1 peptide effective to block said currents.
In a related embodiment, the invention includes a method for selecting compounds for use in treating disorders, such as epilepsy or oversecretion of oxytocin, in which blockade of class E calcium channels is indicated. The selection method includes testing the compound in an assay system that measures class E channel activity and selecting the compound if it blocks calcium currents through such channels at a concentration that is no more than about 50 times a concentration of HG-1 peptide that is effective to block such channels. An exemplary screening assay is the whole-cell patch clamp of neurohypophysial terminals described herein. The selection method may, in addition, utilize additional screens, such as N-, L-, or P/Q-type calcium channel assays, to ensure that the selected compound is selective for class E channels.
In another aspect, the invention includes recombinant methods for producing class E voltage-gated calcium channel blocking peptides. In particular, for use in such a method, the invention includes the nucleotide fragments having the sequences: SEQ ID NOs: 23 to SEQ ID NO: 43.